Plant geranylgeranyl transferases

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a geranylgeranyl transferase subunit. The invention also relates to the construction of a chimeric gene encoding all or a portion of the geranylgeranyl transferase subunit, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the geranylgeranyl transferase subunit in a transformed host cell.

This application claims priority benefit of U.S. Provisional ApplicationSer. No. 60/098,743 filed Sep. 1, 1998, now pending.

FIELD OF THE INVENTION

This invention is in the field of plant molecular biology. Morespecifically, this invention pertains to nucleic acid fragments encodinggeranylgeranyl transferase subunits in plants and seeds.

BACKGROUND OF THE INVENTION

Lipids and proteins associate covalently to form lipid-linked proteinsand noncovalently to form lipoproteins. The lipid portions oflipid-linked proteins anchor their attached proteins to membranes andmediate protein-protein interactions. Proteins form covalent attachmentsto lipids in several ways, one of which is the covalent attachment ofisoprenoid groups, mainly the C₁₅ farnesyl and C₂₀ geranylgeranylresidues.

In mammals, geranylgeranyltransferase is known to catalyze the transferof a geranyl-geranyl moiety from geranylgeranyl pyrophsophate to bothcysteines in Rab proteins (Farnsworth, C. C. etal. (1994)Proc Natl AcadSci USA 91(25):11963-11967) Rab proteins are Ras-related small GTPasesthat are geranylgeranylated on cysteine residues located at or neartheir C termini. Mammalian protein geranylgeranyl transferases types 1and 2 are heterodimers composed of an alpha and beta subunit. The alphasubunit shows homology to the alpha subunits of a closely relatedenzyme, farnesyltransferase.

Farnesyltransferases have been described in pea, tomato, andArabidopsis, but have not been described in monocots. The plantfarnesyltransferases also consist of alpha and beta subunits. Thegeranylgeranyl transferase beta subunit belongs to the proteinprenyltransferase beta subunit family. The beta subunits of the type 1and 2 geranylgeranyltransferases have not been previously described inplants. Work done in yeast has established thatgeranylgeranyltransferases are distinct from the closely relatedfarnesyltransferases.

The mammalian geranylgeranyl transferases require the aid of a RABescort protein (also called component A). RAB escort protein is requiredfor Rab geranylgeranyl transferase activity in mammals. RAB escortprotein binds unprenylated RAB proteins, presents it to the catalyticcomponent B (alpha/beta subunit complex of geranylgeranyltransferase).RAB binding protein remains bound to the prenylated protein after thegeranylgeranyl transfer reaction. Component A may be regenerated bytransferring its prenylated RAB to a protein acceptor.

There is a great deal of interest in identifying the genes that encodegeranylgeranyl transferase in plants. These genes may be used in plantcells to control cell growth. Accordingly, the availability of nucleicacid sequences encoding all or a portion of geranylgeranyl transferaseproteins would facilitate studies to better understand cell growth inplants, provide genetic tools to enhance cell growth in tissue culture,increase the efficiency of gene transfer and help provide more stabletransformations. Geranylgeranyl transferase proteins may also providetargets to facilitate design and/or identification of inhibitors of cellgrowth that may be useful as herbicides.

SUMMARY OF THE INVENTION

The instant invention relates to isolated nucleic acid fragmentsencoding geranylgeranyl transferase subunits. Specifically, thisinvention concerns an isolated nucleic acid fragment encoding ageranylgeranyl transferase type I beta subunit, type II beta subunit orRab escort protein and an isolated nucleic acid fragment that issubstantially similar to an isolated nucleic acid fragment encoding ageranylgeranyl transferase type I beta subunit, type II beta subunit orRab escort protein. In addition, this invention relates to a nucleicacid fragment that is complementary to the nucleic acid fragmentencoding a geranylgeranyl transferase type I beta subunit, type II betasubunit or Rab escort protein.

An additional embodiment of the instant invention pertains to apolypeptide encoding all or a substantial portion of a geranylgeranyltransferase subunit selected from the group consisting of geranylgeranyltransferase type I beta subunit, type II beta subunit and Rab escortprotein.

In another embodiment, the instant invention relates to a chimeric geneencoding a geranylgeranyl transferase type I beta subunit, type II betasubunit or Rab escort protein, or to a chimeric gene that comprises anucleic acid fragment that is complementary to a nucleic acid fragmentencoding a geranylgeranyl transferase type I beta subunit, type II betasubunit or Rab escort protein, operably linked to suitable regulatorysequences, wherein expression of the chimeric gene results in productionof levels of the encoded protein in a transformed host cell that isaltered (i.e., increased or decreased) from the level produced in anuntransformed host cell.

In a further embodiment, the instant invention concerns a transformedhost cell comprising in its genome a chimeric gene encoding ageranylgeranyl transferase type I beta subunit, type II beta subunit orRab escort protein, operably linked to suitable regulatory sequences.Expression of the chimeric gene results in production of altered levelsof the encoded protein in the transformed host cell. The transformedhost cell can be of eukaryotic or prokaryotic origin, and include cellsderived from higher plants and microorganisms. The invention alsoincludes transformed plants that arise from transformed host cells ofhigher plants, and seeds derived from such transformed plants.

An additional embodiment of the instant invention concerns a method ofaltering the level of expression of a geranylgeranyl transferase type Ibeta subunit, type II beta subunit or Rab escort protein in atransformed host cell comprising: a) transforming a host cell with achimeric gene comprising a nucleic acid fragment encoding ageranylgeranyl transferase type I beta subunit, type II beta subunit orRab escort protein; and b) growing the transformed host cell underconditions that are suitable for expression of the chimeric gene whereinexpression of the chimeric gene results in production of altered levelsof geranylgeranyl transferase type I beta subunit, type II beta subunitor Rab escort protein in the transformed host cell.

An addition embodiment of the instant invention concerns a method forobtaining a nucleic acid fragment encoding all or a substantial portionof an amino acid sequence encoding a geranylgeranyl transferase type Ibeta subunit, type II beta subunit or Rab escort protein.

BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS

The invention can be more fully understood from the following detaileddescription and the accompanying Sequence Listing which form a part ofthis application.

Table 1 lists the polypeptides that are described herein, thedesignation of the cDNA clones that comprise the nucleic acid fragmentsencoding polypeptides representing all or a substantial portion of thesepolypeptides, and the corresponding identifier (SEQ ID NO:) as used inthe attached Sequence Listing. The sequence descriptions and SequenceListing attached hereto comply with the rules governing nucleotideand/or amino acid sequence disclosures in patent applications as setforth in 37 C.F.R. §1.821-1.825.

TABLE 1 Geranylgeranyl Transferase Subunits SEQ ID NO: (Nucleo- (AminoProtein Clone Designation tide) Acid) Geranylgeranyl Transferasecco1n.pk066.m2 1 2 Type II Beta Subunit Geranylgeranyl Transferasesf11.pk0074.b7 3 4 Type II Beta Subunit Geranylgeranyl Transferasew1su2.pk0001.h3 5 6 Type II Beta Subunit Geranylgeranyl Transferasesre.pk0040.h8 7 8 Type I Beta Subunit Rab Escort Protein r10n.pk0025.f109 10 Rab Escort Protein wr1.pk0001.c3 11 12

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J 219 (No.2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

In the context of this disclosure, a number of terms shall be utilized.As used herein, a “nucleic acid fragment” is a polymer of RNA or DNAthat is single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases. A nucleic acid fragment in theform of a polymer of DNA may be comprised of one or more segments ofcDNA, genomic DNA or synthetic DNA.

As used herein, “substantially similar” refers to nucleic acid fragmentswherein changes in one or more nucleotide bases results in substitutionof one or more amino acids, but do not affect the functional propertiesof the polypeptide encoded by the nucleotide sequence. “Substantiallysimilar” also refers to nucleic acid fragments wherein changes in one ormore nucleotide bases does not affect the ability of the nucleic acidfragment to mediate alteration of gene expression by gene silencingthrough for example antisense or co-suppression technology.“Substantially similar” also refers to modifications of the nucleic acidfragments of the instant invention such as deletion or insertion of oneor more nucleotides that do not substantially affect the functionalproperties of the resulting transcript vis-{grave over (a)}-vis theability to mediate gene silencing or alteration of the functionalproperties of the resulting protein molecule. It is therefore understoodthat the invention encompasses more than the specific exemplarynucleotide or amino acid sequences and includes functional equivalentsthereof.

For example, it is well known in the art that antisense suppression andco-suppression of gene expression may be accomplished using nucleic acidfragments representing less than the entire coding region of a gene, andby nucleic acid fragments that do not share 100% sequence identity withthe gene to be suppressed. Moreover, alterations in a nucleic acidfragment which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded polypeptide, are well known in the art. Thus, a codon for theamino acid alanine, a hydrophobic amino acid, may be substituted by acodon encoding another less hydrophobic residue, such as glycine, or amore hydrophobic residue, such as valine, leucine, or isoleucine.Similarly, changes which result in substitution of one negativelycharged residue for another, such as aspartic acid for glutamic acid, orone positively charged residue for another, such as lysine for arginine,can also be expected to produce a functionally equivalent product.Nucleotide changes which result in alteration of the N-terminal andC-terminal portions of the polypeptide molecule would also not beexpected to alter the activity of the polypeptide. Each of the proposedmodifications is well within the routine skill in the art, as isdetermination of retention of biological activity of the encodedproducts.

Moreover, substantially similar nucleic acid fragments may also becharacterized by their ability to hybridize. Estimates of such homologyare provided by either DNA-DNA or DNA-RNA hybridization under conditionsof stringency as is well understood by those skilled in the art (Hamesand Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford,U.K.). Stringency conditions can be adjusted to screen for moderatelysimilar fragments, such as homologous sequences from distantly relatedorganisms, to highly similar fragments, such as genes that duplicatefunctional enzymes from closely related organisms. Post-hybridizationwashes determine stringency conditions. One set of preferred conditionsuses a series of washes starting with 6× SSC, 0.5% SDS at roomtemperature for 15 min, then repeated with 2× SSC, 0.5% SDS at 45° C.for 30 min, and then repeated twice with 0.2× SSC, 0.5% SDS at 50° C.for 30 min. A more preferred set of stringent conditions uses highertemperatures in which the washes are identical to those above except forthe temperature of the final two 30 min washes in 0.2× SSC, 0.5% SDS wasincreased to 60° C. Another preferred set of highly stringent conditionsuses two final washes in 0.1× SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant inventionmay also be characterized by the percent identity of the amino acidsequences that they encode to the amino acid sequences disclosed herein,as determined by algorithms commonly employed by those skilled in thisart. Preferred are those nucleic acid fragments whose nucleotidesequences encode amino acid sequences that are 80% identical to theamino acid sequences reported herein. More preferred nucleic acidfragments encode amino acid sequences that are 90% identical to theamino acid sequences reported herein. Most preferred are nucleic acidfragments that encode amino acid sequences that are 95% identical to theamino acid sequences reported herein. Sequence alignments and percentidentity calculations were performed using the Megalign program of theLASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of the sequences was performed using the Clustalmethod of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) withthe default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Defaultparameters for pairwise alignments using the Clustal method were KTUPLE1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequencecomprises an amino acid or a nucleotide sequence that is sufficient toafford putative identification of the protein or gene that the aminoacid or nucleotide sequence comprises. Amino acid and nucleotidesequences can be evaluated either manually by one skilled in the art, orby using computer-based sequence comparison and identification toolsthat employ algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul et al. (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more contiguous nucleotides isnecessary in order to putatively identify a polypeptide or nucleic acidsequence as homologous to a known protein or gene. Moreover, withrespect to nucleotide sequences, gene-specific oligonucleotide probescomprising 30 or more contiguous nucleotides may be used insequence-dependent methods of gene identification (e.g., Southernhybridization) and isolation (e.g., in situ hybridization of bacterialcolonies or bacteriophage plaques). In addition, short oligonucleotidesof 12 or more nucleotides may be used as amplification primers in PCR inorder to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises a nucleotide sequence that will afford specific identificationand/or isolation of a nucleic acid fragment comprising the sequence. Theinstant specification teaches amino acid and nucleotide sequencesencoding polypeptides that comprise one or more particular plantproteins. The skilled artisan, having the benefit of the sequences asreported herein, may now use all or a substantial portion of thedisclosed sequences for purposes known to those skilled in this art.Accordingly, the instant invention comprises the complete sequences asreported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without effecting the amino acidsequence of an encoded polypeptide. Accordingly, the instant inventionrelates to any nucleic acid fragment comprising a nucleotide sequencethat encodes all or a substantial portion of the amino acid sequencesset forth herein. The skilled artisan is well aware of the “codon-bias”exhibited by a specific host cell in usage of nucleotide codons tospecify a given amino acid. Therefore, when synthesizing a nucleic acidfragment for improved expression in a host cell, it is desirable todesign the nucleic acid fragment such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotidebuilding blocks that are chemically synthesized using procedures knownto those skilled in the art. These building blocks are ligated andannealed to form larger nucleic acid fragments which may then beenzymatically assembled to construct the entire desired nucleic acidfragment. “Chemically synthesized”, as related to nucleic acid fragment,means that the component nucleotides were assembled in vitro. Manualchemical synthesis of nucleic acid fragments may be accomplished usingwell established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the nucleic acid fragments can be tailored for optimal geneexpression based on optimization of nucleotide sequence to reflect thecodon bias of the host cell. The skilled artisan appreciates thelikelihood of successful gene expression if codon usage is biasedtowards those codons favored by the host. Determination of preferredcodons can be based on a survey of genes derived from the host cellwhere sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

“Coding sequence” refers to a nucleotide sequence that codes for aspecific amino acid sequence. “Regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. The promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence which can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types, or at different stages ofdevelopment, or in response to different environmental conditions.Promoters which cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed MRNAupstream of the translation start sequence. The translation leadersequence may affect processing of the primary transcript to MRNA, MRNAstability or translation efficiency. Examples of translation leadersequences have been described (Turner and Foster (1995) Mol. Biotechnol.3:225-236).

The “3′ non-coding sequences” refer to nucleotide sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting MRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the MRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al. (1989) PlantCell 1:671-680.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to an RNAtranscript that includes the MRNA and so can be translated into apolypeptide by the cell. “Antisense RNA” refers to an RNA transcriptthat is complementary to all or part of a target primary transcript ormRNA and that blocks the expression of a target gene (see U.S. Pat. No.5,107,065, incorporated herein by reference). The complementarity of anantisense RNA may be with any part of the specific nucleotide sequence,i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, orthe coding sequence. “Functional RNA” refers to sense RNA, antisenseRNA, ribozyme RNA, or other RNA that may not be translated but yet hasan effect on cellular processes.

The term “operably linked” refers to the association of two or morenucleic acid fragments on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). Coding sequencescan be operably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment of the invention. Expression may also refer totranslation of mRNA into a polypeptide. “Antisense inhibition” refers tothe production of antisense RNA transcripts capable of suppressing theexpression of the target protein. “Overexpression” refers to theproduction of a gene product in transgenic organisms that exceeds levelsof production in normal or non-transformed organisms. “Co-suppression”refers to the production of sense RNA transcripts capable of suppressingthe expression of identical or substantially similar foreign orendogenous genes (U.S. Pat. No. 5,231,020, incorporated herein byreference).

“Altered levels” refers to the production of gene product(s) intransgenic organisms in amounts or proportions that differ from that ofnormal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of MRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be but are not limited tointracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere et al. (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein etal. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of severalgeranylgeranyl transferase subunits have been isolated and identified bycomparison of random plant cDNA sequences to public databases containingnucleotide and protein sequences using the BLAST algorithms well knownto those skilled in the art. The nucleic acid fragments of the instantinvention may be used to isolate cDNAs and genes encoding homologousproteins from the same or other plant species. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limitedto, methods of nucleic acid hybridization, and methods of DNA and RNAamplification as exemplified by various uses of nucleic acidamplification technologies (e.g., polymerase chain reaction, ligasechain reaction).

For example, genes encoding other geranylgeranyl transferase type I betasubunit, type II beta subunit or Rab escort protein, either as cDNAs orgenomic DNAs, could be isolated directly by using all or a portion ofthe instant nucleic acid fragments as DNA hybridization probes to screenlibraries from any desired plant employing methodology well known tothose skilled in the art. Specific oligonucleotide probes based upon theinstant nucleic acid sequences can be designed and synthesized bymethods known in the art (Maniatis). Moreover, the entire sequences canbe used directly to synthesize DNA probes by methods known to theskilled artisan such as random primer DNA labeling, nick translation, orend-labeling techniques, or RNA probes using available in vitrotranscription systems. In addition, specific primers can be designed andused to amplify a part or all of the instant sequences. The resultingamplification products can be labeled directly during amplificationreactions or labeled after amplification reactions, and used as probesto isolate full length cDNA or genomic fragments under conditions ofappropriate stringency.

In addition, two short segments of the instant nucleic acid fragmentsmay be used in polymerase chain reaction protocols to amplify longernucleic acid fragments encoding homologous genes from DNA or RNA. Thepolymerase chain reaction may also be performed on a library of clonednucleic acid fragments wherein the sequence of one primer is derivedfrom the instant nucleic acid fragments, and the sequence of the otherprimer takes advantage of the presence of the polyadenylic acid tractsto the 3′ end of the MRNA precursor encoding plant genes. Alternatively,the second primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al. (1988) Proc. Natl. Acad Sci. USA 85:8998-9002)to generate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl.Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220).Products generated by the 3′ and 5′ RACE procedures can be combined togenerate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165).

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of cDNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequences may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen cDNA expression libraries toisolate full-length cDNA clones of interest (Lerner (1984) Adv. Immuno.36:1-34; Maniatis).

The nucleic acid fragments of the instant invention may be used tocreate transgenic plants in which the disclosed polypeptides are presentat higher or lower levels than normal or in cell types or developmentalstages in which they are not normally found. This would have the effectof altering the level of cell growth in those cells.

Overexpression of the proteins of the instant invention may beaccomplished by first constructing a chimeric gene in which the codingregion is operably linked to a promoter capable of directing expressionof a gene in the desired tissues at the desired stage of development.For reasons of convenience, the chimeric gene may comprise promotersequences and translation leader sequences derived from the same genes.3′ Non-coding sequences encoding transcription termination signals mayalso be provided. The instant chimeric gene may also comprise one ormore introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene can then beconstructed. The choice of plasmid vector is dependent upon the methodthat will be used to transform host plants. The skilled artisan is wellaware of the genetic elements that must be present on the plasmid vectorin order to successfully transform, select and propagate host cellscontaining the chimeric gene. The skilled artisan will also recognizethat different independent transformation events will result indifferent levels and patterns of expression (Jones et al. (1985) EMBO J.4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), andthus that multiple events must be screened in order to obtain linesdisplaying the desired expression level and pattern. Such screening maybe accomplished by Southern analysis of DNA, Northern analysis of MRNAexpression, Western analysis of protein expression, or phenotypicanalysis.

For some applications it may be useful to direct the instantpolypeptides to different cellular compartments, or to facilitate itssecretion from the cell. It is thus envisioned that the chimeric genedescribed above may be further supplemented by altering the codingsequence to encode the instant polypeptides with appropriateintracellular targeting sequences such as transit sequences (Keegstra(1989) Cell 56:247-253), signal sequences or sequences encodingendoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. PlantPhys. Plant Mol. Biol. 42:21-53), or nuclear localization signals(Raikhel (1992) Plant Phys.100:1627-1632) added and/or with targetingsequences that are already present removed. While the references citedgive examples of each of these, the list is not exhaustive and moretargeting signals of utility may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genesencoding the instant polypeptides in plants for some applications. Inorder to accomplish this, a chimeric gene designed for co-suppression ofthe instant polypeptide can be constructed by linking a gene or genefragment encoding that polypeptide to plant promoter sequences.Alternatively, a chimeric gene designed to express antisense RNA for allor part of the instant nucleic acid fragment can be constructed bylinking the gene or gene fragment in reverse orientation to plantpromoter sequences. Either the co-suppression or antisense chimericgenes could be introduced into plants via transformation whereinexpression of the corresponding endogenous genes are reduced oreliminated.

Molecular genetic solutions to the generation of plants with alteredgene expression have a decided advantage over more traditional plantbreeding approaches. Changes in plant phenotypes can be produced byspecifically inhibiting expression of one or more genes by antisenseinhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and5,283,323). An antisense or cosuppression construct would act as adominant negative regulator of gene activity. While conventionalmutations can yield negative regulation of gene activity these effectsare most likely recessive. The dominant negative regulation availablewith a transgenic approach may be advantageous from a breedingperspective. In addition, the ability to restrict the expression ofspecific phenotype to the reproductive tissues of the plant by the useof tissue specific promoters may confer agronomic advantages relative toconventional mutations which may have an effect in all tissues in whicha mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations areassociated with the use of antisense or cosuppression technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of sense or antisense genes may require the use ofdifferent chimeric genes utilizing different regulatory elements knownto the skilled artisan. Once transgenic plants are obtained by one ofthe methods described above, it will be necessary to screen individualtransgenics for those that most effectively display the desiredphenotype. Accordingly, the skilled artisan will develop methods forscreening large numbers of transformants. The nature of these screenswill generally be chosen on practical grounds, and is not an inherentpart of the invention. For example, one can screen by looking forchanges in gene expression by using antibodies specific for the proteinencoded by the gene being suppressed, or one could establish assays thatspecifically measure enzyme activity. A preferred method will be onewhich allows large numbers of samples to be processed rapidly, since itwill be expected that a large number of transformants will be negativefor the desired phenotype.

The instant polypeptides (or portions thereof) may be produced inheterologous host cells, particularly in the cells of microbial hosts,and can be used to prepare antibodies to the these proteins by methodswell known to those skilled in the art. The antibodies are useful fordetecting the polypeptides of the instant invention in situ in cells orin vitro in cell extracts. Preferred heterologous host cells forproduction of the instant polypeptides are microbial hosts. Microbialexpression systems and expression vectors containing regulatorysequences that direct high level expression of foreign proteins are wellknown to those skilled in the art. Any of these could be used toconstruct a chimeric gene for production of the instant polypeptides.This chimeric gene could then be introduced into appropriatemicroorganisms via transformation to provide high level expression ofthe encoded geranylgeranyl transferase subunit. An example of a vectorfor high level expression of the instant polypeptides in a bacterialhost is provided (Example 8).

All or a substantial portion of the nucleic acid fragments of theinstant invention may also be used as probes for genetically andphysically mapping the genes that they are a part of, and as markers fortraits linked to those genes. Such information may be useful in plantbreeding in order to develop lines with desired phenotypes. For example,the instant nucleic acid fragments may be used as restriction fragmentlength polymorphism (RFLP) markers. Southern blots (Maniatis) ofrestriction-digested plant genomic DNA may be probed with the nucleicacid fragments of the instant invention. The resulting banding patternsmay then be subjected to genetic analyses using computer programs suchas MapMaker (Lander et al. (1987) Genomics 1:174-181) in order toconstruct a genetic map. In addition, the nucleic acid fragments of theinstant invention may be used to probe Southern blots containingrestriction endonuclease-treated genomic DNAs of a set of individualsrepresenting parent and progeny of a defined genetic cross. Segregationof the DNA polymorphisms is noted and used to calculate the position ofthe instant nucleic acid sequence in the genetic map previously obtainedusing this population (Botstein et al. (1980) Am. J. Hum. Genet.32:314-331).

The production and use of plant gene-derived probes for use in geneticmapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol.Reporter 4:37-4 1. Numerous publications describe genetic mapping ofspecific cDNA clones using the methodology outlined above or variationsthereof. For example, F2 intercross populations, backcross populations,randomly mated populations, near isogenic lines, and other sets ofindividuals may be used for mapping. Such methodologies are well knownto those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences mayalso be used for physical mapping (i.e., placement of sequences onphysical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: APractical Guide, Academic press 1996, pp. 319-346, and references citedtherein). In another embodiment, nucleic acid probes derived from theinstant nucleic acid sequences may be used in direct fluorescence insitu hybridization (FISH) mapping (Trask (1991) Trends Genet.7:149-154). Although current methods of FISH mapping favor use of largeclones (several to several hundred KB; see Laan et al. (1995) GenomeRes. 5:13-20), improvements in sensitivity may allow performance of FISHmapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic andphysical mapping may be carried out using the instant nucleic acidsequences. Examples include allele-specific amplification (Kazazian(1989) J. Lab. Clin. Med 11:95-96), polymorphism of PCR-amplifiedfragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),allele-specific ligation (Landegren et al. (1988) Science241:1077-1080), nucleotide extension reactions (Sokolov (1990) NucleicAcid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat.Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic AcidRes. 1 7:6795-6807). For these methods, the sequence of a nucleic acidfragment is used to design and produce primer pairs for use in theamplification reaction or in primer extension reactions. The design ofsuch primers is well known to those skilled in the art. In methodsemploying PCR-based genetic mapping, it may be necessary to identify DNAsequence differences between the parents of the mapping cross in theregion corresponding to the instant nucleic acid sequence. This,however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instantcDNA clones either by targeted gene disruption protocols or byidentifying specific mutants for these genes contained in a maizepopulation carrying mutations in all possible genes (Ballinger andBenzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995)Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell7:75-84). The latter approach may be accomplished in two ways. First,short segments of the instant nucleic acid fragments may be used inpolymerase chain reaction protocols in conjunction with a mutation tagsequence primer on DNAs prepared from a population of plants in whichMutator transposons or some other mutation-causing DNA element has beenintroduced (see Bensen, supra). The amplification of a specific DNAfragment with these primers indicates the insertion of the mutation tagelement in or near the plant gene encoding the instant polypeptides.Alternatively, the instant nucleic acid fragment may be used as ahybridization probe against PCR amplification products generated fromthe mutation population using the mutation tag sequence primer inconjunction with an arbitrary genomic site primer, such as that for arestriction enzyme site-anchored synthetic adaptor. With either method,a plant containing a mutation in the endogenous gene encoding theinstant polypeptides can be identified and obtained. This mutant plantcan then be used to determine or confirm the natural function of theinstant polypeptides disclosed herein.

EXAMPLES

The present invention is further defined in the following Examples, inwhich all parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these Examples,while indicating preferred embodiments of the invention, are given byway of illustration only. From the above discussion and these Examples,one skilled in the art can ascertain the essential characteristics ofthis invention, and without departing from the spirit and scope thereof,can make various changes and modifications of the invention to adapt itto various usages and conditions.

Example 1 Composition of cDNA Libraries. Isolation and Sequencing ofCDNA Clones

cDNA libraries representing mRNAs from various corn, rice, soybean andwheat tissues were prepared. The characteristics of the libraries aredescribed below.

TABLE 2 cDNA Libraries from Corn, Rice, Soybean and Wheat Library TissueClone cco1n Corn cob of 67 day old plants grown cco1n.pk066.m2 in greenhouse* r10n Rice 15 day old leaf* r10n.pk0025.f10 sf11 Soybean immatureflower sf11.pk0074.b7 sre Soybean root elongation zone 4 to 5 dayssre.pk0040.h8 after germination w1su2 Wheat WLMK8 cDNAs subtracted withw1su2.pk0001.h3 WLM0 cDNAs** wr1 Wheat root from 7 day old seedlingwr1.pk0001.c3 *These libraries were normalized essentially as describedin U.S. Pat. No. 5,482,845, incorporated herein by reference. **WLMK8cDNAs are from wheat seedlings 8 hours after inoculation with Erysiphegraminis f. sp tritici and treatment with6-iodo-2-propoxy-3-propyl-4(3H)-quinazolinone (synthesis and methods ofusing this compound are described in USSN 08/545,827, incorporatedherein by reference). WLMO cDNAs are from wheat seedlings 0 hours afterinoculation with Erysiphe graminis f. sp tritici. Subtraction of thecDNA libraries was achieved using a combination of a cDNA synthesis kit# (Strategene: cat # 200400) and the Clontech PCR-Select cDNASubtraction it (Clontech, cat # PT3138-1) as per manufacturesinstructions.

cDNA libraries may be prepared by any one of many methods available. Forexample, the cDNAs may be introduced into plasmid vectors by firstpreparing the CDNA libraries in Uni-ZAP™ XR vectors according to themanufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.).The Uni-ZAPT™ XR libraries are converted into plasmid librariesaccording to the protocol provided by Stratagene. Upon conversion, cDNAinserts will be contained in the plasmid vector pBluescript. Inaddition, the cDNAs may be introduced directly into precut Bluescript IISK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs),followed by transfection into DH1OB cells according to themanufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts arein plasmid vectors, plasmid DNAs are prepared from randomly pickedbacterial colonies containing recombinant pBluescript plasmids, or theinsert CDNA sequences are amplified via polymerase chain reaction usingprimers specific for vector sequences flanking the inserted cDNAsequences. Amplified insert DNAs or plasmid DNAs are sequenced indye-primer sequencing reactions to generate partial cDNA sequences(expressed sequence tags or “ESTs”; see Adams et al., (1991) Science252:1651-1656). The resulting ESTs are analyzed using a Perkin ElmerModel 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

cDNA clones encoding geranylgeranyl transferase subunits were identifiedby conducting BLAST (Basic Local Alignment Search Tool; Altschul et al.(1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/)searches for similarity to sequences contained in the BLAST “nr”database (comprising all non-redundant GenBank CDS translations,sequences derived from the 3-dimensional structure Brookhaven ProteinData Bank, the last major release of the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The cDNA sequences obtained inExample 1 were analyzed for similarity to all publicly available DNAsequences contained in the “nr” database using the BLASTN algorithmprovided by the National Center for Biotechnology Information (NCBI).The DNA sequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish and States (1993) Nat.Genet. 3:266-272) provided by the NCBI. For convenience, the P-valueprobability) of observing a match of a cDNA sequence to a sequencecontained in the searched databases merely by chance as calculated byBLAST are reported herein as “pLog” values, which represent the negativeof the logarithm of the reported P-value. Accordingly, the greater thepLog value, the greater the likelihood that the cDNA sequence and theBLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding GeranylgeranylTransferase Type II Beta Subunit

The BLASTX search using the EST sequences from clones listed in Table 3revealed similarity of the polypeptides encoded by the cDNAs togeranylgeranyl transferase type II beta subunit from Homo sapiens (NCBIIdentifier No. gi 2506788). Shown in Table 3 are the BLAST results forindividual ESTs (“EST”), the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), or contigs assembled fromtwo or more ESTs (“Contig”):

TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous toHomo sapiens Geranylgeranyl Transferase Type II Beta Subunit CloneStatus BLAST pLog Score to gi 2506788 cco1n.pk066.m2 FIS 106.00sf11.pk0074.b7 FIS 117.00 w1su2.pk0001.h3 FIS  96.70

The data in Table 4 represents a calculation of the percent identity ofthe amino acid sequences set forth in SEQ ID NOs:2, 4 and 6 and the Homosapiens sequence. The percent identity between the amino acid sequencesset forth in SEQ ID NOs:2, 4 and 6 ranges between 71%-82%.

TABLE 4 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toHomo sapiens Geranylgeranyl Transferase Type II Beta Subunit PercentIdentity to SEQ ID NO. gi 2506788 2 11% 4 12% 6 11%

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a geranylgeranyl transferase type IIbeta subunit. These sequences represent the first corn, soybean andwheat sequences encoding geranylgeranyl transferase type II betasubunit.

Example 4 Characterization of cDNA Clones Encoding GeranylgeranylTransferase Type I Beta Subunit

The BLASTX search using the EST sequence from the clone listed in Table5 revealed similarity of the polypeptide encoded by the cDNA togeranylgeranyl transferase type I beta subunit from Arabidopsis thaliana(NCBI Identifier No. gi 3355484). Shown in Table 5 are the BLAST resultsfor individual ESTs (“EST”), the sequences of the entire cDNA insertscomprising the indicated cDNA clones (“FIS”), or contigs assembled fromtwo or more ESTs (“Contig”):

TABLE 5 BLAST Results for Sequences Encoding Polypeptides Homologous toArabidopsis thaliana Geranylgeranyl Transferase Type I Beta SubunitClone Status BLAST pLog Score to gi 3355484 sre.pk0040.h8 FIS 139.00

The data in Table 6 represents a calculation of the percent identity ofthe amino acid sequence set forth in SEQ ID NO:8 and the Arabidopsisthaliana sequence.

TABLE 6 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toArabidopsis thaliana Geranylgeranyl Transferase Type I Beta SubunitPercent Identity to SEQ ID NO. gi 3355484 8 65%

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a geranylgeranyl transferase type I betasubunit. These sequences represent the first soybean sequence encodinggeranylgeranyl transferase type I beta subunit.

Example 5 Characterization of cDNA Clones Encoding Rab Escort Protein

The BLASTX search using the EST sequences from the clones listed inTable 7 revealed similarity of the polypeptides encoded by the cDNAs toRab escort protein from Homo sapiens (NCBI Identifier No. gi 2950156).Shown in Table 7 are the BLAST results for individual ESTs (“EST”), thesequences of the entire cDNA inserts comprising the indicated cDNAclones (“FIS”), or contigs assembled from two or more ESTs (“Contig”):

TABLE 7 BLAST Results for Sequences Encoding Polypeptides Homologous toHomo sapiens Rab Escort Protein Clone Status BLAST pLog Score to gi2950156 r10n.pk0025.f10 FIS 41.04 wr1.pk0001.c3 FIS 29.22

The data in Table 8 represents a calculation of the percent identity ofthe amino acid sequence set forth in SEQ ID NO:9 and 10 and the Homosapiens sequence. The percent identity between the amino acid sequenceset forth in SEQ ID NO:9 and 10 was 76%.

TABLE 8 Percent Identity of Amino Acid Sequences Deduced From theNucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous toHomo sapiens Rab Escort Protein Percent Identity to SEQ ID NO. gi3355484  9 19% 10 21%

Sequence alignments and percent identity calculations were performedusing the Megalign program of the LASERGENE bioinformatics computingsuite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequenceswas performed using the Clustal method of alignment (Higgins and Sharp(1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10,GAP LENGTH PENALTY=10). Default parameters for pairwise alignments usingthe Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5. Sequence alignments and BLAST scores and probabilities indicatethat the nucleic acid fragments comprising the instant cDNA clonesencode a substantial portion of a Rab escort protein. These sequencesrepresent the first rice and wheat sequences encoding Rab escortproteins.

Example 6 Expression of Chimeric Genes in Monocot Cells

A chimeric gene comprising a cDNA encoding the instant polypeptides insense orientation with respect to the maize 27 kD zein promoter that islocated 5′ to the cDNA fragment, and the 10 kD zein 3′ end that islocated 3′ to the cDNA fragment, can be constructed. The cDNA fragmentof this gene may be generated by polymerase chain reaction (PCR) of thecDNA clone using appropriate oligonucleotide primers. Cloning sites(NcoI or Smal) can be incorporated into the oligonucleotides to provideproper orientation of the DNA fragment when inserted into the digestedvector pML 103 as described below. Amplification is then performed in astandard PCR. The amplified DNA is then digested with restrictionenzymes NcoI and SmaI and fractionated on an agarose gel. Theappropriate band can be isolated from the gel and combined with a 4.9 kbNcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has beendeposited under the terms of the Budapest Treaty at ATCC (American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110-2209),and bears accession number ATCC 97366. The DNA segment from pML103contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zeingene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kDzein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA canbe ligated at 15° C. overnight, essentially as described (Maniatis). Theligated DNA may then be used to transform E. coli XL 1-Blue (EpicurianColi XL-1 Blue™; Stratagene). Bacterial transformants can be screened byrestriction enzyme digestion of plasmid DNA and limited nucleotidesequence analysis using the dideoxy chain termination method (Sequenase™DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid constructwould comprise a chimeric gene encoding, in the 5′ to 3′ direction, themaize 27 kD zein promoter, a cDNA fragment encoding the instantpolypeptides, and the 10 kD zein 3′ region.

The chimeric gene described above can then be introduced into corn cellsby the following procedure. Immature corn embryos can be dissected fromdeveloping caryopses derived from crosses of the inbred corn lines H99and LH132. The embryos are isolated 10 to 11 days after pollination whenthey are 1.0 to 1.5 mm long. The embryos are then placed with theaxis-side facing down and in contact with agarose-solidified N6 medium(Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept inthe dark at 27° C. Friable embryogenic callus consisting ofundifferentiated masses of cells with somatic proembryoids and embryoidsborne on suspensor structures proliferates from the scutellum of theseimmature embryos. The embryogenic callus isolated from the primaryexplant can be cultured on N6 medium and sub-cultured on this mediumevery 2 to 3 weeks.

The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag,Frankfurt, Germany) may be used in transformation experiments in orderto provide for a selectable marker. This plasmid contains the Pat gene(see European Patent Publication 0 242 236) which encodesphosphinothricin acetyl transferase (PAT). The enzyme PAT confersresistance to herbicidal glutamine synthetase inhibitors such asphosphinothricin. The pat gene in p35S/Ac is under the control of the35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature313:810-812) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

The particle bombardment method (Klein et al. (1987) Nature 327:70-73)may be used to transfer genes to the callus culture cells. According tothis method, gold particles (1 μm in diameter) are coated with DNA usingthe following technique. Ten μg of plasmid DNAs are added to 50 μL of asuspension of gold particles (60 mg per mL). Calcium chloride (50 μL ofa 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution)are added to the particles. The suspension is vortexed during theaddition of these solutions. After 10 minutes, the tubes are brieflycentrifuged (5 sec at 15,000 rpm) and the supernatant removed. Theparticles are resuspended in 200 μL of absolute ethanol, centrifugedagain and the supernatant removed. The ethanol rinse is performed againand the particles resuspended in a final volume of 30 μL of ethanol. Analiquot (5 μL) of the DNA-coated particles are then accelerated into thecorn tissue with a Biolistic™ PDS-1 000/He (Bio-Rad gold particles canbe placed in the center of a Kapton™ flying disc (Bio-Rad Labs). TheInstruments, Hercules Calif.), using a helium pressure of 1000 psi, agap distance of 0.5 cm and a flying distance of 1.0 cm.

For bombardment, the embryogenic tissue is placed on filter paper overagarose-solidified N6 medium. The tissue is arranged as a thin lawn andcovered a circular area of about 5 cm in diameter. The petri dishcontaining the tissue can be placed in the chamber of the PDS-1000/Heapproximately 8 cm from the stopping screen. The air in the chamber isthen evacuated to a vacuum of 28 inches of Hg. The macrocarrier isaccelerated with a helium shock wave using a rupture membrane thatbursts when the He pressure in the shock tube reaches 1000 psi.

Seven days after bombardment the tissue can be transferred to N6 mediumthat contains gluphosinate (2 mg per liter) and lacks casein or proline.The tissue continues to grow slowly on this medium. After an additional2 weeks the tissue can be transferred to fresh N6 medium containinggluphosinate. After 6 weeks, areas of about 1 cm in diameter of activelygrowing callus can be identified on some of the plates containing theglufosinate-supplemented medium. These calli may continue to grow whensub-cultured on the selective medium.

Plants can be regenerated from the transgenic callus by firsttransferring clusters of tissue to N6 medium supplemented with 0.2 mgper liter of 2,4-D. After two weeks the tissue can be transferred toregeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 7 Expression of Chimeric Genes in Dicot Cells

A seed-specific expression cassette composed of the promoter andtranscription terminator from the gene encoding the β subunit of theseed storage protein phaseolin from the bean Phaseolus vulgaris (Doyleet al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expressionof the instant polypeptides in transformed soybean. The phaseolincassette includes about 500 nucleotides upstream (5′) from thetranslation initiation codon and about 1650 nucleotides downstream (3′)from the translation stop codon of phaseolin. Between the 5′ and 3′regions are the unique restriction endonuclease sites Nco I (whichincludes the ATG translation initiation codon), SmaI, KpnI and XbaI. Theentire cassette is flanked by Hind III sites.

The cDNA fragment of this gene may be generated by polymerase chainreaction (PCR) of the cDNA clone using appropriate oligonucleotideprimers. Cloning sites can be incorporated into the oligonucleotides toprovide proper orientation of the DNA fragment when inserted into theexpression vector. Amplification is then performed as described above,and the isolated fragment is inserted into a pUC18 vector carrying theseed expression cassette.

Soybean embroys may then be transformed with the expression vectorcomprising sequences encoding the instant polypeptides. To inducesomatic embryos, cotyledons, 3-5 mm in length dissected from surfacesterilized, immature seeds of the soybean cultivar A2872, can becultured in the light or dark at 26° C. on an appropriate agar mediumfor 6-10 weeks. Somatic embryos which produce secondary embryos are thenexcised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos which multiplied as early,globular staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can maintained in 35 mL liquidmedia on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a16:8 hour day/night schedule. Cultures are subcultured every two weeksby inoculating approximately 35 mg of tissue into 35 mL of liquidmedium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein et al. (1987) Nature (London)327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS 1000/HEinstrument (helium retrofit) can be used for these transformations.

A selectable marker gene which can be used to facilitate soybeantransformation is a chimeric gene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The seed expression cassette comprising the phaseolin 5′region, the fragment encoding the instant polypeptides and the phaseolin3′ region can be isolated as a restriction fragment. This fragment canthen be inserted into a unique restriction site of the vector carryingthe marker gene.

To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μL 70% ethanol andresuspended in 40 μL of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five μL of theDNA-coated gold particles are then loaded on each macro carrier disk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi and the chamber is evacuated to a vacuum of 28 inchesmercury. The tissue is placed approximately 3.5 inches away from theretaining screen and bombarded three times. Following bombardment, thetissue can be divided in half and placed back into liquid and culturedas described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media, and eleven to twelve days post bombardment with freshmedia containing 50 mg/mL hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

EXAMPLE 8 Expression of Chimeric Genes in Microbial Cells

The cDNAs encoding the instant polypeptides can be inserted into the T7E. coli expression vector pBT430. This vector is a derivative of pET-3a(Rosenberg et al. (1987) Gene 56:125-135) which employs thebacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 wasconstructed by first destroying the EcoR I and Hind III sites in pET-3aat their original positions. An oligonucleotide adaptor containing EcoRI and Hind III sites was inserted at the BamH I site of pET-3a. Thiscreated pET-3aM with additional unique cloning sites for insertion ofgenes into the expression vector. Then, the Nde I site at the positionof translation initiation was converted to an Nco I site usingoligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM inthis region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

Plasmid DNA containing a cDNA may be appropriately digested to release anucleic acid fragment encoding the protein. This fragment may then bepurified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer andagarose contain 10 μg/ml ethidium bromide for visualization of the DNAfragment. The fragment can then be purified from the agarose gel bydigestion with GELase™ (Epicentre Technologies) according to themanufacturer's instructions, ethanol precipitated, dried and resuspendedin 20 μL of water. Appropriate oligonucleotide adapters may be ligatedto the fragment using T4 DNA ligase (New England Biolabs, Beverly,Mass.). The fragment containing the ligated adapters can be purifiedfrom the excess adapters using low melting agarose as described above.The vector pBT430 is digested, dephosphorylated with alkalinephosphatase (NEB) and deproteinized with phenol/chloroform as describedabove. The prepared vector pBT430 and fragment can then be ligated at16° C. for 15 hours followed by transformation into DH5 electrocompetentcells (GIBCO BRL). Transformants can be selected on agar platescontaining LB media and 100 μg/mL ampicillin. Transformants containingthe gene encoding the instant polypeptides are then screened for thecorrect orientation with respect to the T7 promoter by restrictionenzyme analysis.

For high level expression, a plasmid clone with the cDNA insert in thecorrect orientation relative to the T7 promoter can be transformed intoE. coli strain BL21(DE3) (Studier et al. (1986) J. Mol. Biol.189:113-130). Cultures are grown in LB medium containing ampicillin (100mg/L) at 25° C. At an optical density at 600 mn of approximately 1, IPTG(isopropylthio-β-galactoside, the inducer) can be added to a finalconcentration of 0.4 mM and incubation can be continued for 3 h at 25°.Cells are then harvested by centrifugation and re-suspended in 50 μL of50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenylmethylsulfonyl fluoride. A small amount of 1 mm glass beads can be addedand the mixture sonicated 3 times for about 5 seconds each time with amicroprobe sonicator. The mixture is centrifuged and the proteinconcentration of the supernatant determined. One μg of protein from thesoluble fraction of the culture can be separated by SDS-polyacrylamidegel electrophoresis. Gels can be observed for protein bands migrating atthe expected molecular weight.

12 1 1411 DNA Zea mays unsure (785) unsure (792) 1 gcacgagata ttttgaaatagcccccgcca cgaaattctt cagccatcgt ggaggggttc 60 agttcagggg aggactcgactcaggaaaca ggagcacgag cggagcagaa ggcagtcatg 120 gcggatgagg tggagcttgctgcggaccag cacgtccgct acatcgtcac ggtggagaag 180 aagaaggact cctttgagtcgctggtgatg gagcacatcc gcctcaacgg cgcctactgg 240 ggcctcacca cgctcgacctcctccacaag ctccatgccg tagatgccgc cgaggtcgtc 300 gactggatca tgtcctgctaccaccccgaa tctggtggat ttggagggaa cgttgggcat 360 gacccgcatg tcctctacacgcttagcgcc gtgcaggtcc tctgcctttt cgatcggctc 420 gatgtccttg acgtcgacaaggttgctgat tatgtcgccg gactgcaaaa caaggatgga 480 tcattttctg gcgatatttggggtgaagtt gacactaggt tctcgtatat tgccttatgt 540 accttatcat tactgcaccgtctgcataag attgatgtgc aaaaagctgt ggatttcgtt 600 gttagctgta agaacttggatggcggattt ggagctatgc caggagggga gtctcatgct 660 ggacaaatat tttgttgtgtcggcgcgctc gcaatcaccg ggtccctgca tcacattgat 720 agagacctcc tcggatggtggctctgtgag cgccagtgta aagacggagg acttaatggg 780 cggcntgaga anctagctgatgtggtttgc tactcgtggt gggtgctatc gagcctagtc 840 atgattgaca gagtgcattggattgacaag gaaaagctaa cgaaattcat actgaactgt 900 caggacaaag agaacggcggcatttcagat agaccagata atgcagtcga tatctatcac 960 acgtactttg gaattgcagggctttcatta atggagtacc ccggggtgaa gcctttggat 1020 cctgcctatg cactaccattgcacgttgtc aatcggattt tcttgaaaaa atagaacatt 1080 ccattcgatc tgcggcgcagagatatgctg agatggcgct gtcagatgtg gacgctactt 1140 cacagaacca cgattcgacggagaaatagc tgagggggaa tcaattacag gaacatgttg 1200 ggataccata atcttggacttgtattctcg cattggctgt ggccgcttag tcgatgtttt 1260 tttgtcattt ggcacttgccctgttgaatg cttggtgcat gctgtgtttt gctagttgat 1320 ctgattctct tgtttaagtcgtaacattgt gtgtcctgat gacgacgaca ttagtcagga 1380 gctatctata aacagtatgtcttttttgaa a 1411 2 318 PRT Zea mays UNSURE (227) 2 Met Ala Asp Glu ValGlu Leu Ala Ala Asp Gln His Val Arg Tyr Ile 1 5 10 15 Val Thr Val GluLys Lys Lys Asp Ser Phe Glu Ser Leu Val Met Glu 20 25 30 His Ile Arg LeuAsn Gly Ala Tyr Trp Gly Leu Thr Thr Leu Asp Leu 35 40 45 Leu His Lys LeuHis Ala Val Asp Ala Ala Glu Val Val Asp Trp Ile 50 55 60 Met Ser Cys TyrHis Pro Glu Ser Gly Gly Phe Gly Gly Asn Val Gly 65 70 75 80 His Asp ProHis Val Leu Tyr Thr Leu Ser Ala Val Gln Val Leu Cys 85 90 95 Leu Phe AspArg Leu Asp Val Leu Asp Val Asp Lys Val Ala Asp Tyr 100 105 110 Val AlaGly Leu Gln Asn Lys Asp Gly Ser Phe Ser Gly Asp Ile Trp 115 120 125 GlyGlu Val Asp Thr Arg Phe Ser Tyr Ile Ala Leu Cys Thr Leu Ser 130 135 140Leu Leu His Arg Leu His Lys Ile Asp Val Gln Lys Ala Val Asp Phe 145 150155 160 Val Val Ser Cys Lys Asn Leu Asp Gly Gly Phe Gly Ala Met Pro Gly165 170 175 Gly Glu Ser His Ala Gly Gln Ile Phe Cys Cys Val Gly Ala LeuAla 180 185 190 Ile Thr Gly Ser Leu His His Ile Asp Arg Asp Leu Leu GlyTrp Trp 195 200 205 Leu Cys Glu Arg Gln Cys Lys Asp Gly Gly Leu Asn GlyArg Leu Arg 210 215 220 Thr Ser Xaa Cys Glu Val Cys Tyr Ser Trp Trp ValLeu Ser Ser Leu 225 230 235 240 Val Met Ile Asp Arg Val His Trp Ile AspLys Glu Lys Leu Thr Lys 245 250 255 Phe Ile Leu Asn Cys Gln Asp Lys GluAsn Gly Gly Ile Ser Asp Arg 260 265 270 Pro Asp Asn Ala Val Asp Ile TyrHis Thr Tyr Phe Gly Ile Ala Gly 275 280 285 Leu Ser Leu Met Glu Tyr ProGly Val Lys Pro Leu Asp Pro Ala Tyr 290 295 300 Ala Leu Pro Leu His ValVal Asn Arg Ile Phe Leu Lys Lys 305 310 315 3 1306 DNA Glycine max 3gcacgagttt cgcttgacct tggcacaaac taacagagca ttagcgtttc tacttcgtat 60cactgccgcg acccttctgg attccgacgg tgactttgat taaggagtcc gatgggagag 120ctggccactg agaaacatgt tcgatatata ttatcagttg aaaagaggaa agataacttt 180gaatctgtcg taatggagca tctaagaatg aatggggcat attggggatt gaccactctg 240gatcttctag gaaagcttca taccgtcgat gttgatgagg ttgtttcgtg gttgatgagt 300tgtcagcatg actcaggggg atttggtgga aatgttggac atgatccgca catcctctat 360acactaagtg ctgtgcaggt gttgtctctc tttgataagc tggatgttat tgatgtagat 420aaggtcacaa gttatattgt cagcctgcaa aatgaagatg gatccttttc aggggatatg 480tggggtgaag ttgatacacg gttctcatat attgctattt gttgtctatc aatattacat 540cgcttggata aaatcaatgt ggagaaggct gtgaagtaca ttataagttg caaaaatatg 600gatggtggtt ttgggtgcac tcctggtggg gaatctcatg ctggtcaaat tttctgttgt 660gtgggggccc ttgccataac agggtcacta gatcttgttg acaaagacct acttggttgg 720tggttatgcg agcgacaggt taaatctgga ggtctgaatg ggcgtcctga gaaacatcct 780gatgtctgct actcatggtg ggttctttct agcctgatca tgattgatag ggtacattgg 840attagtaagg agaagcttat aaagttcatc ttagactgcc aggacacaga aaatggtgga 900atttcggaca ggccagatga tgctgtggat gtctttcata cattctttgg ggtggctgga 960ctttctcttc ttgaatatcc agggctgaaa ccagtagatc cagcttatgc tttacctgtt 1020gatgttgtaa atagaattat ttttactaaa taaggacttt agtagttaag ttcgatgata 1080attttccagt aatgacaaaa tcttgggttt gtaagactca ctgttgggag ttggaccccc 1140tcctcccatc cccagcccaa aaacagttaa tttcttaaaa acagtgttaa cattttgagc 1200ttcttttagt taaattgctg tggtacgaca tgtaaagatt gatcagtatt gtagtcaacc 1260atcaaattta tgctactagt tactacataa aaaaaaaaaa aaaaaa 1306 4 313 PRTGlycine max 4 Met Gly Glu Leu Ala Thr Glu Lys His Val Arg Tyr Ile LeuSer Val 1 5 10 15 Glu Lys Arg Lys Asp Asn Phe Glu Ser Val Val Met GluHis Leu Arg 20 25 30 Met Asn Gly Ala Tyr Trp Gly Leu Thr Thr Leu Asp LeuLeu Gly Lys 35 40 45 Leu His Thr Val Asp Val Asp Glu Val Val Ser Trp LeuMet Ser Cys 50 55 60 Gln His Asp Ser Gly Gly Phe Gly Gly Asn Val Gly HisAsp Pro His 65 70 75 80 Ile Leu Tyr Thr Leu Ser Ala Val Gln Val Leu SerLeu Phe Asp Lys 85 90 95 Leu Asp Val Ile Asp Val Asp Lys Val Thr Ser TyrIle Val Ser Leu 100 105 110 Gln Asn Glu Asp Gly Ser Phe Ser Gly Asp MetTrp Gly Glu Val Asp 115 120 125 Thr Arg Phe Ser Tyr Ile Ala Ile Cys CysLeu Ser Ile Leu His Arg 130 135 140 Leu Asp Lys Ile Asn Val Glu Lys AlaVal Lys Tyr Ile Ile Ser Cys 145 150 155 160 Lys Asn Met Asp Gly Gly PheGly Cys Thr Pro Gly Gly Glu Ser His 165 170 175 Ala Gly Gln Ile Phe CysCys Val Gly Ala Leu Ala Ile Thr Gly Ser 180 185 190 Leu Asp Leu Val AspLys Asp Leu Leu Gly Trp Trp Leu Cys Glu Arg 195 200 205 Gln Val Lys SerGly Gly Leu Asn Gly Arg Pro Glu Lys His Pro Asp 210 215 220 Val Cys TyrSer Trp Trp Val Leu Ser Ser Leu Ile Met Ile Asp Arg 225 230 235 240 ValHis Trp Ile Ser Lys Glu Lys Leu Ile Lys Phe Ile Leu Asp Cys 245 250 255Gln Asp Thr Glu Asn Gly Gly Ile Ser Asp Arg Pro Asp Asp Ala Val 260 265270 Asp Val Phe His Thr Phe Phe Gly Val Ala Gly Leu Ser Leu Leu Glu 275280 285 Tyr Pro Gly Leu Lys Pro Val Asp Pro Ala Tyr Ala Leu Pro Val Asp290 295 300 Val Val Asn Arg Ile Ile Phe Thr Lys 305 310 5 1230 DNATriticum aestivum 5 tcaagctatg catccaacgc gttgggagct ctcccatatggtcgacctgc aggcggccgc 60 gaattcacta gtgattagcg tggtcgcggc cgaggtcgtcgactggatca tgtcgtgcta 120 ccacccggaa tctggtgggt tcggggggaa cgtggggcacgacccgcatg tcctctacac 180 gctcagcacc gtgcaggtcc tctgcctctt cgatcggctcgatgttcttg atgcagacaa 240 aattgctgat tatattactg gacttcagaa tgaggatggatcattttctg gtgatatttg 300 gggtgaagtt gatactaggt tctcttatat ttccatatgcaccttgtcat tactgcatcg 360 tctgcacaaa attaatgtgg acaaggctgt agaatatattgttagctgta agaacttgga 420 cggcgggttt ggagcgatgc cgggagggga gtctcatgctgggcagatat tctgttgtgt 480 tggtgctctc gcaatcaccg gctctttgca tcacattgatagagatctcc ttggatggtg 540 gctttgtgag cgccagtgta gagatggggg gctcaatgggcgtcctgaga aacttgctga 600 tgtgtgctac tcatggtggg tgttatcaag cttgataattattgatagag tgcactggat 660 tgacaaggaa aaacttgcaa agttcatatt gaactgtcaggacaaggaaa atggtggaat 720 ttcagataga ccagataatg cggtcgatat ctaccacacgtactttggag ttgcagggct 780 ctcattgatg gagtatcctg gagtgaagcc tatggatcctgcctacgccc tccctttaga 840 tgttgtcaac aggatcttct tgacaaaaca acaatagtgtgccttagcta ggaagatcat 900 gttgtaacgg cgttgacgtc aggtcagcac gagtggagagcttacccctc cttcggtagc 960 tcgcgctgat gtttctgatc actcccatgc atgagatcatggctttgaac gctcgatgat 1020 atagtgcaga cctcatattt accaggaaat ccggacacttgttatgtaga agagtgtaac 1080 gtccaaggac tgagaatcaa attcaatcaa gattattcttgttggaaaaa aaaaaaaaaa 1140 aaaaaaaaaa aaaaactcga cctgcccggg cggccgctcgaaatcgaatt cccgcggccg 1200 ccatggcggc cgggagcatg cgacgtcggg 1230 6 291PRT Triticum aestivum 6 Gln Ala Met His Pro Thr Arg Trp Glu Leu Ser HisMet Val Asp Leu 1 5 10 15 Gln Ala Ala Ala Asn Ser Leu Val Ile Ser ValVal Ala Ala Glu Val 20 25 30 Val Asp Trp Ile Met Ser Cys Tyr His Pro GluSer Gly Gly Phe Gly 35 40 45 Gly Asn Val Gly His Asp Pro His Val Leu TyrThr Leu Ser Thr Val 50 55 60 Gln Val Leu Cys Leu Phe Asp Arg Leu Asp ValLeu Asp Ala Asp Lys 65 70 75 80 Ile Ala Asp Tyr Ile Thr Gly Leu Gln AsnGlu Asp Gly Ser Phe Ser 85 90 95 Gly Asp Ile Trp Gly Glu Val Asp Thr ArgPhe Ser Tyr Ile Ser Ile 100 105 110 Cys Thr Leu Ser Leu Leu His Arg LeuHis Lys Ile Asn Val Asp Lys 115 120 125 Ala Val Glu Tyr Ile Val Ser CysLys Asn Leu Asp Gly Gly Phe Gly 130 135 140 Ala Met Pro Gly Gly Glu SerHis Ala Gly Gln Ile Phe Cys Cys Val 145 150 155 160 Gly Ala Leu Ala IleThr Gly Ser Leu His His Ile Asp Arg Asp Leu 165 170 175 Leu Gly Trp TrpLeu Cys Glu Arg Gln Cys Arg Asp Gly Gly Leu Asn 180 185 190 Gly Arg ProGlu Lys Leu Ala Asp Val Cys Tyr Ser Trp Trp Val Leu 195 200 205 Ser SerLeu Ile Ile Ile Asp Arg Val His Trp Ile Asp Lys Glu Lys 210 215 220 LeuAla Lys Phe Ile Leu Asn Cys Gln Asp Lys Glu Asn Gly Gly Ile 225 230 235240 Ser Asp Arg Pro Asp Asn Ala Val Asp Ile Tyr His Thr Tyr Phe Gly 245250 255 Val Ala Gly Leu Ser Leu Met Glu Tyr Pro Gly Val Lys Pro Met Asp260 265 270 Pro Ala Tyr Ala Leu Pro Leu Asp Val Val Asn Arg Ile Phe LeuThr 275 280 285 Lys Gln Gln 290 7 1422 DNA Glycine max 7 gcacgaggttctgattctga tttctcacta gtaaaattga ggatcggcat tcagcaatga 60 tgaattccacagaagaagag tatcgtggct gcgagattat ggagaaagat gttcatgtca 120 cgtttctcgagttaatgtac tatttactcc catctccgta cgagtcccaa gagatcaacc 180 atctcactctcgcttacttt gtcatctctg gacttgacat cctcgactct ctccacaaag 240 ttgcgaaggatgctgttgtc agttgggttt tgtccttcca agctcacccc ggtgccaaga 300 ctgatctcaatgatgggcaa ttctatggct ttcatggatc caaaacttca cagtttcctc 360 cagatgagaatggggttttg attcacaaca acagtcactt ggcaagtact tattgtgcca 420 tttccatattgaaaattgtt ggttatgaat tgtccaatct tgactctgaa acaattgtga 480 cttctatgaggaaccttcaa cagcctgatg gaagtttcat tccgattcat actggaggcg 540 aaacagatcttaggtttgtg tattgtgcag ctgccatctg tttcatgttg gataactgga 600 gtggcatggacaaggagaaa accaaggatt acatattacg ttgccagtct tatgatggtg 660 gctttggattagttcctggt gcagaatcgc atggaggtgc aacttattgt gctatggcat 720 ctctccgattaatgggattc attgaagata atattctctc aagttgtgct tcatcttctt 780 tgatagatgcgccattgctg ctggactgga tcttgcagag gcagggaact gatgggggtt 840 ttcaaggtagaccaaataaa tctagcgata catgttatgc attttggatt ggagccgttt 900 taaggattttggggggcttc aaatttgttg acaataaggc tctacgtgga tttttgcttt 960 cttgtcaatataagtatggt ggtttcagca aattccctgg ggagtatcca gacctatacc 1020 actcctactatggattcact gctttcagcc tgttggaaga atctggcttg aaatcacttt 1080 tttcggaactgggaatcact gaaaatgctg cactggcact ctagcttaga ttcagaaatg 1140 gatgtacctttatacctgac atttcttaca tattatatga acctctcagc actaatccac 1200 tcttactggacttttttttt tattctttca caaatttagg tggagtgtaa attttccatt 1260 tcatttgattgtatttgtgt gcattaattc aggtaattgg acctcttcta tttagaacaa 1320 gctttttttgtattgctttc tttttgtttt atttacattt cgatgaagtt tattattgaa 1380 tatgttaatttgaagttcag cataaaaaaa aaaaaaaaaa aa 1422 8 355 PRT Glycine max 8 Met MetAsn Ser Thr Glu Glu Glu Tyr Arg Gly Cys Glu Ile Met Glu 1 5 10 15 LysAsp Val His Val Thr Phe Leu Glu Leu Met Tyr Tyr Leu Leu Pro 20 25 30 SerPro Tyr Glu Ser Gln Glu Ile Asn His Leu Thr Leu Ala Tyr Phe 35 40 45 ValIle Ser Gly Leu Asp Ile Leu Asp Ser Leu His Lys Val Ala Lys 50 55 60 AspAla Val Val Ser Trp Val Leu Ser Phe Gln Ala His Pro Gly Ala 65 70 75 80Lys Thr Asp Leu Asn Asp Gly Gln Phe Tyr Gly Phe His Gly Ser Lys 85 90 95Thr Ser Gln Phe Pro Pro Asp Glu Asn Gly Val Leu Ile His Asn Asn 100 105110 Ser His Leu Ala Ser Thr Tyr Cys Ala Ile Ser Ile Leu Lys Ile Val 115120 125 Gly Tyr Glu Leu Ser Asn Leu Asp Ser Glu Thr Ile Val Thr Ser Met130 135 140 Arg Asn Leu Gln Gln Pro Asp Gly Ser Phe Ile Pro Ile His ThrGly 145 150 155 160 Gly Glu Thr Asp Leu Arg Phe Val Tyr Cys Ala Ala AlaIle Cys Phe 165 170 175 Met Leu Asp Asn Trp Ser Gly Met Asp Lys Glu LysThr Lys Asp Tyr 180 185 190 Ile Leu Arg Cys Gln Ser Tyr Asp Gly Gly PheGly Leu Val Pro Gly 195 200 205 Ala Glu Ser His Gly Gly Ala Thr Tyr CysAla Met Ala Ser Leu Arg 210 215 220 Leu Met Gly Phe Ile Glu Asp Asn IleLeu Ser Ser Cys Ala Ser Ser 225 230 235 240 Ser Leu Ile Asp Ala Pro LeuLeu Leu Asp Trp Ile Leu Gln Arg Gln 245 250 255 Gly Thr Asp Gly Gly PheGln Gly Arg Pro Asn Lys Ser Ser Asp Thr 260 265 270 Cys Tyr Ala Phe TrpIle Gly Ala Val Leu Arg Ile Leu Gly Gly Phe 275 280 285 Lys Phe Val AspAsn Lys Ala Leu Arg Gly Phe Leu Leu Ser Cys Gln 290 295 300 Tyr Lys TyrGly Gly Phe Ser Lys Phe Pro Gly Glu Tyr Pro Asp Leu 305 310 315 320 TyrHis Ser Tyr Tyr Gly Phe Thr Ala Phe Ser Leu Leu Glu Glu Ser 325 330 335Gly Leu Lys Ser Leu Phe Ser Glu Leu Gly Ile Thr Glu Asn Ala Ala 340 345350 Leu Ala Leu 355 9 2335 DNA Oryza sativa 9 gcacgagctt acacggcagcgtgcgcagga ggagaaaatt gcggagggga tccacagttc 60 cagccgcgtg tgacggcggcgtgcggttgc cgcggcgtgc tgctcgagcc gctttacgac 120 tgatccgagc ggctttttccggcgatcatg gcggacgcgc ccgccaccgg cggcggattc 180 cccgcgcagg actaccccaccatcgacccc acctcgttcg acgtggtcct ctgcggcacc 240 ggcctcccgg agtccgtcctcgccgccgcc tgcgccgccg ccgggaagac ggtcctccac 300 gtcgacccca accctttctacggctccctc ttctcctccc tccctctccc ttccctcccc 360 tccttcctct ccccctccccctccgacgac cccgccccct ccccttcccc ctcctccgcc 420 gccgccgtcg atctccgccgccgcagcccg tactcggagg tggagacctc gggggcggtg 480 cccgagccgt ccaggcgcttcaccgccgac ctggtgggcc ccaggctgct ctactgcgcc 540 gacgaggccg tcgacctcctcctcaggtca gggggaagcc accatgtgga gttcaagagc 600 gtggaggggg gaaccctcctctactgggac ggcgatctct acccggtgcc ggactcgagg 660 caggccatct tcaaggacaccaccctccag ctcagggaga agaacctact cttcaggttc 720 ttcaagcttg tgcaggcccacattgccgcg tcggctgccg gcgccgccgc ggcgggggaa 780 ggcgaggcct ccggtaggctgcccgatgag gacctggacc tccccttcgt cgaattcctc 840 aagaggcaga atctttcgcccaagatgaga gcggttgtct tgtatgcaat tgccatggcg 900 gattatgatc aggatggtgtggagtcttgt gagaggttgt taacaacgag agagggagtc 960 aagacaattg ctctttactcctcatctatt gggaggtttg ctaatgcaga gggggctttc 1020 atttatccta tgtatgggcatggtgagctg cctcaagctt tctgccgctg tgctgctgtt 1080 aaaggtgccc tatatgtattgcgaatgcca gccacagcac ttcttgttga tgaggaaaaa 1140 aagcgttatg taggtatcagattggcatct ggtcaggata ttttgtgcca acagttgata 1200 ctcgatccat catatgaaattccttccttg gatatgccaa gtgatgcacc agtatcaaat 1260 ttgccaagaa aagttgctaggggaatatgc ataatcagca gctctgtgag acaagataca 1320 tcaaatgttc tggttgttttccccccaaag tcactagaag aggagcaaat tactgctgtt 1380 cgggtgcttc agttgagcagcaatttagca gtatgccctc ctggaatgtt catggcatat 1440 ctctctactc cctgtactgatgccttcact ggaaagaaat gcatcagcaa agcaatagat 1500 gctcttttct caactaaggtttctaatgat ttggaagatc atttggagaa aaacagtgaa 1560 gaaaataagg agagtgtgaagccaacccta ctctggagct gtgtgtatgt acaagagatt 1620 atacagggaa catctggtactgcattgtca tgccccatac ctgatgaaaa tatggactac 1680 aggagtatac ttgaatcaacaaaaatgctg ttcactgata tttgtcctaa tgaagagttc 1740 ctgcctagaa attcagctcccaaatatgct tctgataatg actctgattc tgcagagtaa 1800 atccaaactt gcaaagggctttgtattcta catgacaggg ttatcgttga agatatctgt 1860 tccatattac agtgttccccaaagtcacgg taagaaatat tgcagcaagg gggagccctt 1920 tacgttggtg agatttgccaggtttctctt tgactagatt gcagatgcct acattgttcc 1980 atcattggta cgacatattatgatactggt aaaacactca agagagagcg atgcagatgt 2040 ccttggcatt ttgacatttggagaacatca taccacatga accgcttagt gtgattacta 2100 catccatatg gttctctgaattctatgctg aatttgttgg cacataggag tactgtacta 2160 cataggaagg atactgctgagttctatgct aaatctgtgt atttttggtg ggctgcctga 2220 atcattgtac aagcgaacgacagaaatgga tatcaaatga ggtccctccg agagggagtt 2280 gcattacatg catccttatcgatctgcgcg gtcaaaaaaa aaaaaaaaaa aaaaa 2335 10 550 PRT Oryza sativa 10Met Ala Asp Ala Pro Ala Thr Gly Gly Gly Phe Pro Ala Gln Asp Tyr 1 5 1015 Pro Thr Ile Asp Pro Thr Ser Phe Asp Val Val Leu Cys Gly Thr Gly 20 2530 Leu Pro Glu Ser Val Leu Ala Ala Ala Cys Ala Ala Ala Gly Lys Thr 35 4045 Val Leu His Val Asp Pro Asn Pro Phe Tyr Gly Ser Leu Phe Ser Ser 50 5560 Leu Pro Leu Pro Ser Leu Pro Ser Phe Leu Ser Pro Ser Pro Ser Asp 65 7075 80 Asp Pro Ala Pro Ser Pro Ser Pro Ser Ser Ala Ala Ala Val Asp Leu 8590 95 Arg Arg Arg Ser Pro Tyr Ser Glu Val Glu Thr Ser Gly Ala Val Pro100 105 110 Glu Pro Ser Arg Arg Phe Thr Ala Asp Leu Val Gly Pro Arg LeuLeu 115 120 125 Tyr Cys Ala Asp Glu Ala Val Asp Leu Leu Leu Arg Ser GlyGly Ser 130 135 140 His His Val Glu Phe Lys Ser Val Glu Gly Gly Thr LeuLeu Tyr Trp 145 150 155 160 Asp Gly Asp Leu Tyr Pro Val Pro Asp Ser ArgGln Ala Ile Phe Lys 165 170 175 Asp Thr Thr Leu Gln Leu Arg Glu Lys AsnLeu Leu Phe Arg Phe Phe 180 185 190 Lys Leu Val Gln Ala His Ile Ala AlaSer Ala Ala Gly Ala Ala Ala 195 200 205 Ala Gly Glu Gly Glu Ala Ser GlyArg Leu Pro Asp Glu Asp Leu Asp 210 215 220 Leu Pro Phe Val Glu Phe LeuLys Arg Gln Asn Leu Ser Pro Lys Met 225 230 235 240 Arg Ala Val Val LeuTyr Ala Ile Ala Met Ala Asp Tyr Asp Gln Asp 245 250 255 Gly Val Glu SerCys Glu Arg Leu Leu Thr Thr Arg Glu Gly Val Lys 260 265 270 Thr Ile AlaLeu Tyr Ser Ser Ser Ile Gly Arg Phe Ala Asn Ala Glu 275 280 285 Gly AlaPhe Ile Tyr Pro Met Tyr Gly His Gly Glu Leu Pro Gln Ala 290 295 300 PheCys Arg Cys Ala Ala Val Lys Gly Ala Leu Tyr Val Leu Arg Met 305 310 315320 Pro Ala Thr Ala Leu Leu Val Asp Glu Glu Lys Lys Arg Tyr Val Gly 325330 335 Ile Arg Leu Ala Ser Gly Gln Asp Ile Leu Cys Gln Gln Leu Ile Leu340 345 350 Asp Pro Ser Tyr Glu Ile Pro Ser Leu Asp Met Pro Ser Asp AlaPro 355 360 365 Val Ser Asn Leu Pro Arg Lys Val Ala Arg Gly Ile Cys IleIle Ser 370 375 380 Ser Ser Val Arg Gln Asp Thr Ser Asn Val Leu Val ValPhe Pro Pro 385 390 395 400 Lys Ser Leu Glu Glu Glu Gln Ile Thr Ala ValArg Val Leu Gln Leu 405 410 415 Ser Ser Asn Leu Ala Val Cys Pro Pro GlyMet Phe Met Ala Tyr Leu 420 425 430 Ser Thr Pro Cys Thr Asp Ala Phe ThrGly Lys Lys Cys Ile Ser Lys 435 440 445 Ala Ile Asp Ala Leu Phe Ser ThrLys Val Ser Asn Asp Leu Glu Asp 450 455 460 His Leu Glu Lys Asn Ser GluGlu Asn Lys Glu Ser Val Lys Pro Thr 465 470 475 480 Leu Leu Trp Ser CysVal Tyr Val Gln Glu Ile Ile Gln Gly Thr Ser 485 490 495 Gly Thr Ala LeuSer Cys Pro Ile Pro Asp Glu Asn Met Asp Tyr Arg 500 505 510 Ser Ile LeuGlu Ser Thr Lys Met Leu Phe Thr Asp Ile Cys Pro Asn 515 520 525 Glu GluPhe Leu Pro Arg Asn Ser Ala Pro Lys Tyr Ala Ser Asp Asn 530 535 540 AspSer Asp Ser Ala Glu 545 550 11 1359 DNA Triticum aestivum 11 gcacgagaggacttggacct cccctttatt gaattcctca agaaacagca gcttcaaccc 60 aagattagagcggttgtgct atatgcaatt gccatggcag attatgatca agatgccgca 120 gacacatgcgagaaattgct aacaacaaga gatggaatca agaccctagc tctttattcc 180 tcgtccattgggaggtttgc taatgcccaa ggtgctttca tttatcctat gtacgggcat 240 ggtgagctactgcaagcttt ctgtcgcttt gctgctgtta agggtgccct atatgtgttg 300 cggatgccagtcacagccct cctcgtggac caggaaaaga agcgttatat aggcaccaga 360 ttggcttctggtcaggatat tttgtgccag cagttgatac ttggtccctc gtacaaaatt 420 ccttcattggacatgccatc tgatgcttca gactcaaact tgacgagaaa agttgccagg 480 ggagtatgcataatcagcag ctccataaaa gaggcttcat caaatgttct ggttgttttc 540 cccccaaaatcattacaaga gcagcaagct acagctgttc gggcgcttca gctgagcagc 600 aatgtagcagtatgccctcc tggaatgttc atggtatatc tgtctactcc ctgtactgat 660 gcctttacgggaaagcagta cataaacaag gcaatggagg ttcttttcag tactcaggct 720 tcagatgattcagaaggcca tttggagaca accagcaaaa acatcgagga tagaaagcca 780 gtgctaatctggagttgtgt gtatgttcaa gagatcacac agggaacatc tggtgctgta 840 ttgtcatgccccatgccgga tgaaaacctg gactacagag atatactgga atcgacaaaa 900 cagttatttacagatattta tcctgacgaa gaattcctgc ctagaaacgc aactcctaaa 960 tatgccgacgatgactctga tcttgcagag tagaaacatg tttgcagagg gttagtgttt 1020 ttctggacaaatattccaca gaagatacat gaaggcattt tagatacatc gcaccaacat 1080 ggatgatctgctcagtgagc gagaccacag acaacgttga tggttgtatg gcttgttgca 1140 gtgcctgtagttattactgc acaagctaca tgtggcgcct ttcgtttgtt gcactggttc 1200 ttgctacatgtggcgccttt catttgttgc actggttctt tgtacgagtg aaacagagta 1260 atgaaattgagtttaaacta cttgtctgat gcaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1320 aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1359 12 330 PRT Triticum aestivum 12 AlaArg Glu Asp Leu Asp Leu Pro Phe Ile Glu Phe Leu Lys Lys Gln 1 5 10 15Gln Leu Gln Pro Lys Ile Arg Ala Val Val Leu Tyr Ala Ile Ala Met 20 25 30Ala Asp Tyr Asp Gln Asp Ala Ala Asp Thr Cys Glu Lys Leu Leu Thr 35 40 45Thr Arg Asp Gly Ile Lys Thr Leu Ala Leu Tyr Ser Ser Ser Ile Gly 50 55 60Arg Phe Ala Asn Ala Gln Gly Ala Phe Ile Tyr Pro Met Tyr Gly His 65 70 7580 Gly Glu Leu Leu Gln Ala Phe Cys Arg Phe Ala Ala Val Lys Gly Ala 85 9095 Leu Tyr Val Leu Arg Met Pro Val Thr Ala Leu Leu Val Asp Gln Glu 100105 110 Lys Lys Arg Tyr Ile Gly Thr Arg Leu Ala Ser Gly Gln Asp Ile Leu115 120 125 Cys Gln Gln Leu Ile Leu Gly Pro Ser Tyr Lys Ile Pro Ser LeuAsp 130 135 140 Met Pro Ser Asp Ala Ser Asp Ser Asn Leu Thr Arg Lys ValAla Arg 145 150 155 160 Gly Val Cys Ile Ile Ser Ser Ser Ile Lys Glu AlaSer Ser Asn Val 165 170 175 Leu Val Val Phe Pro Pro Lys Ser Leu Gln GluGln Gln Ala Thr Ala 180 185 190 Val Arg Ala Leu Gln Leu Ser Ser Asn ValAla Val Cys Pro Pro Gly 195 200 205 Met Phe Met Val Tyr Leu Ser Thr ProCys Thr Asp Ala Phe Thr Gly 210 215 220 Lys Gln Tyr Ile Asn Lys Ala MetGlu Val Leu Phe Ser Thr Gln Ala 225 230 235 240 Ser Asp Asp Ser Glu GlyHis Leu Glu Thr Thr Ser Lys Asn Ile Glu 245 250 255 Asp Arg Lys Pro ValLeu Ile Trp Ser Cys Val Tyr Val Gln Glu Ile 260 265 270 Thr Gln Gly ThrSer Gly Ala Val Leu Ser Cys Pro Met Pro Asp Glu 275 280 285 Asn Leu AspTyr Arg Asp Ile Leu Glu Ser Thr Lys Gln Leu Phe Thr 290 295 300 Asp IleTyr Pro Asp Glu Glu Phe Leu Pro Arg Asn Ala Thr Pro Lys 305 310 315 320Tyr Ala Asp Asp Asp Ser Asp Leu Ala Glu 325 330

What is claimed is:
 1. An isolated nucleic acid fragment encoding ageranylgeranyl transferase type II beta subunit comprising a memberselected from the group consisting of: (a) an isolated nucleic acidfragment encoding an amino acid sequence of at least 290 amino acidsthat is at least 70% identical to the amino acid sequence set forth in amember selected from the group consisting of SEQ ID NO:2, 4 and 6; (b)an isolated nucleic acid fragment that is complementary to (a).
 2. Theisolated nucleic acid fragment of claim 1 wherein the nucleic acidfragment is a functional RNA.
 3. The isolated nucleic acid fragment ofclaim 1 wherein the nucleotide sequence of the fragment comprises thesequence set forth in a member selected from the group consisting of SEQID NO: 1, 3 and
 5. 4. A chimeric gene comprising the nucleic acidfragment of claim 1 operably linked to suitable regulatory sequences. 5.A transformed host cell comprising the chimeric gene of claim
 4. 6. Anisolated nucleic acid fragment encoding a geranylgeranyl transferasetype I beta subunit comprising a member selected from the groupconsisting of: (a) an isolated nucleic acid fragment encoding an aminoacid sequence of at least 350 amino acids that is at least 70% identicalto the amino acid sequence set forth in SEQ ID NO:8; (b) an isolatednucleic acid fragment that is complementary to (a).
 7. The isolatednucleic acid fragment of claim 6 wherein the nucleic acid fragment is afunctional RNA.
 8. The isolated nucleic acid fragment of claim 6 whereinthe nucleotide sequence of the fragment comprises the sequence set forthin SEQ ID NO:7.
 9. A chimeric gene comprising the nucleic acid fragmentof claim 6 operably linked to suitable regulatory sequences.
 10. Atransformed host cell comprising the chimeric gene of claim 9.